John G. Torrey

Isolation and Cultivation in vitro of the Actinomycete Causing Root Nodulation in Comptonia

The soil actinomycete causing formation of nitrogen-fixing symbiotic nodules on roots of the woody angiosperm Comptonia peregrina (L.) Coult. (Myricaceae) has been isolated from surface-sterilized root nodules after incubation and enzyme maceration. The filamentous bacterium grows slowly in pure culture on a yeast extract medium, producing sporogenous bodies which form large numbers of ovoid spores. Reinfection of sand-grown or aeroponically grown seedlings of Comptonia was achieved repeatedly with inocula prepared from suspensions of the Comptonia isolate. The same actinomycete has been reisolated from these seedling nodules. The induced nodules are highly active in the acetylene-reduction assay, and plants grow vigorously without an exogenous supply of fixed nitrogen.

Growth kinetics and nitrogenase induction in Frankia sp. HFPArI 3 grown in batch culture

Kinetics of growth and nitrogenase induction in Frankia sp. ArI3 were studied in batch culture. Growth on defined medium with NH 4 + as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH 4 + arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 mM C2H4 · mg protein−1 · min−1) was sufficient to support growth on N2 and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K2 HPO4/KH2PO4, 0.1 mM CaCl2 and MgSO4. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10µg protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO2 and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.

A comparison of cultural characteristics and infectivity of Frankia isolates from root nodules of Casuarina species

The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.

Nitrogen Fixation by Actinomycete-Nodulated Angiosperms

Nitrogen fixation by symbiotic associations between soil bacteria belonging to the actinomycetes and root systems of a diversified group of woody dicotyledonous plants is less generally well known than that by the legume-Rhizobium symbiosis. The fixation of dinitrogen by nodulated legumes is a major mechanism for entry of reduced nitrogen into agricultural lands and, to a much lesser extent, to wooded ecosystems. For forested areas, woodlands, wetlands, and fields, nodulated plants like the alders (Alnus), bog plants like sweet gale (Myrica gale), and roadside and disturbed area invaders such as sweet fern

Endogenous and exogenous influences on the regulation of lateral root formation

According to Sutton and Tinus [1] who adopted the definition by Russell [2], a lateral root is a side root ‘that arises by cell division in the pericycle of the parent root. The resulting dome of tissue penetrates the cortex. When the lateral root emerges from the parent root, its apical meristem is comparable with that of the apex of the parent root.’

Root Hair Infection in Actinomycete-Induced Root Nodule Initiation in Casuarina, Myrica, and Comptonia

The infection process leading to the development of root nodules of Comptonia peregrina, Casuarina cunning hamiana, Myrica gale, and M cerifera was studied by light and electron microscopy. Deformed growth of root hairs was observed as early as 24 h after seedlings grown aeroponically or hydroponically were inoculated with suspensions of crushed nodules or cultures of the actinomycetous endophyte of Comptonia The extent of root hair deformation showed a positive correlation with the number of nodules which subsequently developed The essential features of infection in each of these species were very similar. The actinomycete entered a deformed root hair of the host in a region of folding of the cell wall A convoluted elaboration of the root hair wall which occurred at this presumptive penetration site was continuous with the more evenly deposited capsule of the endophytic actinomycete An associated feature of this wall deposition was thickening of the cell wall of the infected root hair and the adjacent prenodular cells. The actinomycete encapsulation was thickest at the presumed site of penetration and thinner in later stages of endophytic growth away from this site. These observations suggest a period of initial disequilibrium caused by the infection, followed by more harmonious symbiotic growth The observation of a morphologically and cytologically similar root hair infection process in these three genera indicates that root hair infection involves a specific and orderly interaction which represents the common mode of invasion in the initiation of actinomycete-induced root nodules

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

SummaryThe first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.

Immunochemical Analysis of Relationships Among Isolated Frankiae (Actinomycetales)

An immunological study of nine strains of pure-cultured members of the order Actinomycetales isolated from symbiotic root nodules was undertaken to establish the taxonomic relationships of these organisms. Based on the data from a simple double-diffusion (Ouchterlony) precipitation procedure, these isolates, which tentatively have been classified in the genus Frankia (family Frankiaceae), could be divided into two major groups. The first group, designated serotype I, included all of the Actinomycetales strains isolated from Alnus, Comptonia, and Myrica host plants. The second group, serotype II, included Actinomycetales strains isolated from Elaeagnus host plants. No ambiguous cross-reactions with unrelated members of the Actinomycetales were observed. Crushed nodule suspensions cross-reacted in the same manner as pure-cultured frankiae, suggesting that the technique could be important in screening for novel, naturally occurring strains. A comparative study in which an immunofluorescence procedure was used failed to show reliable correlations among the Actinomycetales strains tested. We concluded that the immunodiffusion technique was the better of the two methods for Frankia strain characterization.

Ultrastructure of freeze-substitutedFrankia strain HFPCcI 3, the actinomycete isolated from root nodules ofCasuarina cunninghamiana

SummaryFrankia strain HFPCcI 3 is an actinomycete isolated from root nodules ofCasuarina cunninghamiana. In culture it exhibits typicalFrankia morphology and may produce three distinct morphological forms: branching septate hyphae, terminal or intercalary sporangia, and specialized structures termed vesicles which are the purported site of nitrogenase activity. An examination of the ultrastructure of all three morphological forms using both conventional chemical fixation (CF) and quick-freezing followed by freeze-substitution (FS) reveals some interesting differences between the two fixation methods. Unique to FS material are: 1. smooth membrane profiles; 2. lack of mesosomes; 3. lack of discernible nucleoid regions with condensed chromatin; 4. clarity of cytoplasmic elements such as ribosomes and granular bodies; 5. large cytoplasmic tubules in hyphae and young sporangia; 6. outer wall layer not widely separated from the spherical portion of the vesicle, and 7. bundles of microfilaments in vesicles. The quality of preservation after FS appears to be far superior to that obtained with CF. Accordingly the structures observed after FS are thought to represent more faithfully the structure of the living cell.

Studies of an effective strain of Frankia from Allocasuarina lehmanniana of the Casuarinaceae

SummarySeedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.